Thyroid cancer is the most common malignancy of endocrine tissues and most patients require lifelong surveillance with diagnostic thyroid-stimulating hormone (TSH)-stimulated radioiodine uptake as well as remnant or metastatic ablation with radioidine therapy. The PI, who was both co- inventor and co-developer with Genzyme of wild-type recombinant human (rh) TSH currently approved for diagnosis, now proposes to develop more potent and more efficacious second generation analogs of rh TSH for the 10-20% of poorly responsive patients. Our previous structure- function studies resulted in the discovery of the first analogs of TSH and other glycoprotein hormones with major increases in receptor binding affinity and bioactivity ("superactive analogs"). In the current proposal we plan to produce, purify and characterize large amounts of such novel TSH analogs for commercial development. Since TSH in vivo bioassays require higher quantities of hormones than that obtainable from transient transfection experiments, we propose in Aim 1 to develop stable cell lines producing high levels of candidate superactive analogs and produce quantities sufficient for initial animal studies. This Aim will consist of construction of dicistronic vectors with amplifiable markers and mutated subunit cDNAs, amplification, production and concentration of unpurified analogs. Aim 2 will consist of development of a novel, high capacity analog purification method suitable for commercial scale-up. Aim 3 will consist of rigorous quantification and characterization of selected purified analogs by multiple physicochemical methods, including carbohydrate analysis. In Aim 4 analogs will be examined in the now classic mouse rh TSH in vivo bioassay first developed by the PI, with full dose response curves to assess increased potency and maximal efficacy of TSH analogs in order to select a lead candidate for further commercial development. [unreadable] [unreadable] [unreadable]